Project Summary: The hypothalamic pituitary adrenal (HPA) axis is hyperactive in many patients with major depressive disorder (MOD). This dysfunction normalizes with successful treatment and may be a commonality behind treatments. Novel treatments should target the source of HPA axis hyperactivity: presumably dysregulated corticotropin releasing factor (CRF) from the paraventricular hypothalamus (PVN). PVN activity is regulated by the coordination of numerous limbic structures and plasticity in one or more of these regions may cause enhanced PVN CRF output and subsequent HPA axis dysfunction. One likely culprit is the central amygdala (CeA), another region expressing high concentrations of CRF and thought to mediate stress- induced behavior. The Aims of the present research are to determine (1) if, in addition to behavioral effects, chronic increased CRF drive from the CeA reproduces endocrine changes associated with depressive symptoms and (2) if chronically overexpressing CRF in the CeA results in gene expression changes in PVN CRF neurons that correlate with the observed endocrine and behavioral disruptions. A lentiviral vector will overexpress CRF in CRF-expressing neurons (LVCRFp3.OCRF) in the CeA of adult animals. CRF-expressing cells will be easily identifiable by creating a transgenic mouse in which CRF- expressing cells also express green fluorescent protein (GFP). These cells can be isolated via fluorescence activated cell sorting and subjected to gene expression analysis to compare the CRF and non CRF- expressing cells in the PVN and CeA under basal conditions or after chronically overexpressing CRF in the CeA. It is expected that chronic CeA CRF overexpression will result in HPA axis hyperactivity that can be explained at least partially by gene expression changes in PVN CRF cells. Relevance to Public Health: Chronic elevations in CRF may initiate a chain of events altering gene expression and endocrine signaling and eventually leading to symptoms of depression. CRF is released by numerous brain structures but the relationship between CRF in these regions is incompletely understood because previous technology has been unable to mimic chronic CRF elevations. Using superior techniques, CRF will be overexpressed in one region and then CRF cells in another region will be isolated to identify gene-expression changes that may correlate with observed endocrine and behavioral alterations. [unreadable] [unreadable]